The present invention relates to single-chain antibodies against hepatitis B virus core protein, genes thereof, and therapeutic agents for hepatitis B using the same. In particular, the present invention relates to single-chain antibodies characterized in that they inhibit DNA synthesis of hepatitis B virus by binding to the core protein of said virus, DNAs encoding said single-chain antibodies, vectors comprising said DNAs, transformants transformed with said vectors, a process for producing said single-chain antibodies, and therapeutic agents for hepatitis B using such single-chain antibodies or genes thereof.
In Japan, hepatitis B virus (hereinafter sometimes abbreviated as HBV) accounts for a large part of the etiology of chronic hepatitis, as well as hepatitis C virus. Although the proportion of the virus carriers tends to decrease as a result of the recent preventive treatment by vaccination, there still exists a substantial number of patients with chronic hepatitis B, and medical treatments for this disease is quite important in terms of prophylaxis of primary hepatic cancer.
As medical treatments for patients with chronic hepatitis B, those treatments that aim to exclude the virus by eliciting the host immunity, such as the interferon therapy, the steroid withdrawal therapy, and the propagermanium therapy, or the antiviral agent therapies (adenine arabinoside monophosphate, hereinafter sometimes abbreviated as ara-AMP), Lamivudine) are now being used. In the interferon therapy, however, a long-term clinical improvement cannot be observed, although a transient decrease in amount of serological markers of viral activities occurs. Likewise, although ara-AMP has been used with some effect, it is hard to say that the results are satisfactory at present. A treatment in which interferon and ara-AMP are combined and alternately administered exhibits neurotoxicity, and therefore has problems in its use. Furthermore, fulminant hepatitis B, which is one of the forms of acute hepatitis B, is a disease causing a high lethality, and there exist no effective therapeutic agents for this disease presently. Thus, although there is a strong need for developing an anti-hepatitis B virus therapy based on a different mechanism from those of conventional methods, no useful therapies have not yet been developed.
As one of antiviral therapies, there is a method using antibodies specific for viral antigens. Although a method that intracellularly expresses such an antibody may possibly inhibit only the target antigen without damaging the host cells by virtue of the high specificity of the antibody, when IgGs are administered from outside of the cells, they can not inhibit the functions of the viral antigens existing within the cells due to the low efficiency of uptake into the cells. Furthermore, when genes for the H and L strands of an anti-viral antigen-IgG are introduced into cells that are not natural antibody forming cells, the H and L strands expressed from the transgenes will not necessarily form disulfide bonds as efficient as in antibody producing cells to produce IgG in its active form.
On the other hand, a protein produced by expressing a contiguous sequence of cDNAs, in which one cDNA corresponding to the variable region derived from the L strand of an antibody (hereinafter sometimes abbreviated as VL) and another cDNA corresponding to the variable region derived from the H strand of the antibody (hereinafter sometimes abbreviated as VH) are connected together through an appropriate oligonucleotide linker encoding a highly flexible peptide sequence, can specifically bind to the antigen while taking a structure containing no disulfide bonds. Such proteins are called xe2x80x9csingle-chain antibodiesxe2x80x9d, and characterized in that they do not require to be expressed in antibody producing cells in order to form their proper three-dimensional structures as in the above-mentioned IgG, and even when expressed in cells other than antibody producing cells, they have activity of specifically binding to the antigen (McCafferty J. et al., Nature (London), 348, 552-554 (1990); and Marks J. D. et al., J. Mol. Biol. 222, 581-597 (1991)).
As examples of researches on single-chain antibodies aiming at their application to diseases, for example, cancer, those studies may be mentioned in which (a) cDNAs for a single-chain antibody and for a physiologically active substance such as a toxin are ligated together and expressed with the expectation that they will attack and thereby reduce the target cells, or in which (b) the specific binding of the single-chain antibody to the antigen is expected in itself to produce a desired effect.
As specific examples of the above (a), studies have been reported on conjugates in which TGF-xcex1 or a toxin such as the diphtheria toxin is coupled to an anti-EGFR single-chain antibody (Schmidt M. and Wels W., Br. J. Cancer, 74, 853-862 (1996)), anti-erbB-2 single-chain antibody (Wels W. et al., Cancer Res., 52, 6310-6317 (1992); and Schmidt M. and Wels W., Br. J. Cancer, 74,853-862 (1996)), and anti-CD 40 single-chain antibody (Francesco J A. et al., Cancer Res., 55, 3099-3104 (1995)), or on conjugates in which a neurotoxin derived from eosinophil is coupled to an anti-transferrin receptor single-chain antibody (Newton D L. et al., J. Biol. Chem., 269, 26739-26745 (1994)). In these studies, conjugates between a toxin and a single-chain antibody were prepared and the single-chain antibodies were allowed to specifically bind, for example, to target cells with the expectation that only the target cells bound by the single-chain antibodies will be necrotized by the toxin.
As specific examples of the above (b), studies have been reported on single-chain antibodies against erbB-2 (Grim J. et al., Am. J. Respir. Cell. Mol. Biol., 15, 348-354 (1996); Jannot C. B. et al., Oncogene, 13, 275-282 (1996), EGFR (Jannot C B. et al., Oncogene, 13, 275-282 (1996)), and Ras (Werge T M. et al., FEBS Lett., 351, 393-396 (1994)). Furthermore, MMLV engineered so that it expresses an anti-MHC single-chain antibody on its surface has been used in an application for directly delivering it to MHC.
Examples of reported studies on single-chain antibodies against viruses aiming at their application to diseases are those studies against HIV (Wu Y. et al., J. Virol., 70, 3290-3297 (1996); Levy-Mintz P., J. Virol., 70, 8821-8832 (1996); Mhashilkar A M. et al., EMBO J., 14, 1542-1551 (1995)), Foot-and-mouth disease virus (Mason P. et al., Virology, 224, 548-554 (1996)), and Tick-borne fravivirus (Jiang W., J. Virol., 69, 1044-1049 (1995)). In these studies, the targets of single-chain antibodies against HIV are nonstructural proteins such as Rev (Wu Y. et al., J. Virol., 70, 3290-3297 (1996)) and Tat (Mhashilkar A M. et al., EMBO J., 14, 1542-1551 (1995)). Since Rev protein binds to HIV-1 mRNA containing RRE expressed in HIV-1 infected cells and promotes expression of gag, pol, and env gene, binding of a single-chain antibody to this protein can inactivate Rev, and thereby suppress propagation of the virus by reduced expression of gag, pol, and env gene.
Tat protein has a transactivator activity, and specific binding of a single-chain antibody to this protein inhibits this activity and thereby suppresses propagation of HIV. In the case of the single-chain antibody against Tick-borne fravivirus, of which target is a viral surface antigen, it has a neutralizing activity and its binding to the structural protein on the viral surface decreases the viral infection and the synthesis of viral proteins.
However, all the antigens to which these single-chain antibodies are targeted are nonstructural proteins or structural proteins on the viral surface. A drawback of viral surface antigens is that the target antigens are prone to undergo mutation due to strong immuno-pressure exerted thereon, and the specific binding of single-chain antibody is thereby disturbed. For example, there exists a region prone to undergo mutation, called hypervariable region, and the region may be a cause that inhibits the specific binding of the single-chain antibody. Likewise, non-structural proteins may undergo mutations, even though the frequency is lower than that of viral surface antigens, and therefore they have a drawback that the binding of single-chain antibody is thereby similarly disturbed.
On the other hand, the only known example of single-chain antibody against HBV is a single-chain antibody against a surface antigen of HBV (Biotechniques 18:832 (1995), WO95/33832). However, since this single-chain antibody is against a surface antigen as described above, it has a drawback that the specific binding is inhibited by mutations produced in the target antigen. Furthermore, the above single-chain antibody against HBV is produced merely for the purpose of applying it to purification or immunoassay of the antigen.
Thus, the present invention has been made in the light of the above conventional problems, and it aims to provide single-chain antibodies against hepatitis B virus core protein, genes thereof, and therapeutic agents for hepatitis B using the same. More particularly, the present invention aims to provide single-chain antibodies characterized in that they suppress DNA synthesis of said virus by binding to hepatitis B virus core protein, DNAs encoding said single-chain antibodies, vectors containing said DNAs, transformants transformed with said vectors, a process for producing the above single-chain antibodies, and therapeutic agents for hepatitis B using such single-chain antibodies or genes thereof.
By noting the fact that propagation of hepatitis B virus proceeds through a process characteristic of hepadnavirus, the present inventors have developed a novel method for suppressing propagation of hepatitis B virus as a means of solving the above-described problems.
It is known that replication of hepatitis B virus proceeds through a process as described below.
Within virions in the blood, the viral gene exists as a circular incompletely duplex DNA having about 10% single-stranded parts. After infection, when transferred into the nuclei, however, the single-stranded parts are repaired to give a closed circular double-stranded DNA, and the mRNAs for viral proteins and pregenome RNA are transcribed by cellular RNA polymerase from the four open reading frames. Among these substances, the pregenome RNA is packaged together with hepatitis B virus DNA polymerase and reverse transcriptase into the core particle consisting of the core protein, covered by coat protein after synthesis of the minus strand and partial synthesis of the plus strand in the core particle by reverse transcription, and the mature hepatitis B virions are then released out of the cells.
It is believed that the core protein plays an important role in the process of reverse transcription from the pregenome RNA and that the core protein is essential for the virus to have a structure capable of being packaged (Ganem, D., Hepadxc3x8naviridae and their replication, In xe2x80x9cFields Virologyxe2x80x9d (B. N. Fields, D. M. Knipe and P. M. Howley, Eds.-in-Chief) 3rd ed., 2703-2737 (1996), Lippincott-Raven Publishers, Philadelphia; Hirsh, R. et al., Nature, 344, 552-555 (1990); and Lavinem J. et al., J. Virol., 63, 4257-4263 (1989)). It is therefore expected that if one can inhibit functions of the core protein, the viral maturation process subsequent to the reverse transcription, which occurs in the capsid, may be thereby suppressed.
Since the capsid exists in the cells, an agent inhibiting functions of the core protein has to exist within the cells. As methods for inhibiting functions of the core protein within the cells, those methods may be contemplated in which an anti-core protein antibody is intracellularly expressed or in which a mutant core protein is expressed.
The present inventors have immunized a mouse with hepatitis B virus core protein, purified RNA from the spleen, prepared cDNA fragments that encode polypeptides corresponding to VL and VH regions of an antibody against hepatitis B virus core protein by PCR method, and then obtained a single-chain antibody against the core protein (abbreviated as anti-core protein single-chain antibody) to complete the present invention.
In addition, the present inventors have introduced the above gene into a mammalian expression vector in order to express the anti-core protein single-chain antibody in human-derived cultured cells that persistently produce hepatitis B virus, and thereby succeeded in observing significant decrease in amount of replicative intermediate DNAs for hepatitis B virus, that is, suppression of propagation of hepatitis B virus, although the amount of mRNA for hepatitis B virus transcribed within the transformed cells was not affected when compared to that of mRNA in the negative control transformant. The present inventors have thus arrived at the present invention.
Accordingly, the gist of the present invention relates to:
(1) DNA characterized in that it encodes a single-chain antibody capable of binding to hepatitis B virus core protein;
(2) DNA of the above (1) which comprises a DNA selected from the group consisting of:
(A) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4,
(B) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 4,
(C) DNA which comprises the base sequence shown in SEQ ID NO: 3,
(D) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 3, and
(E) DNA which hybridizes under stringent conditions to DNA set forth in any one of (A) to (D);
(3) DNA of the above (1) which comprises a DNA selected from the group consisting of:
(F) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6,
(G) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 6,
(H) DNA which comprises the base sequence shown in SEQ ID NO: 5,
(I) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5, and
(J) DNA which hybridizes under stringent conditions to DNA set forth in any one of (F) to (I);
(4) DNA of the above (1) which comprises both of a DNA selected from the group consisting of the following (A) to (E) and a DNA selected from the group consisting of the following (F) to (J):
(A) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4,
(B) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 4,
(C) DNA which comprises the base sequence shown in SEQ ID NO: 3,
(D) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 3,
(E) DNA which hybridizes under stringent conditions to DNA set forth in any one of (A) to (D),
(F) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6,
(G) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 6,
(H) DNA which comprises the base sequence shown in SEQ ID NO: 5,
(I) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5, and
(J) DNA which hybridizes under stringent conditions to DNA set forth in any one of (F) to (I);
(5) DNA of the above (1) which comprises a DNA selected from the group consisting of:
(K) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2,
(L) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2,
(M) DNA which comprises the base sequence shown in SEQ ID NO: 1,
(N) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 1, and
(O) DNA which hybridizes under stringent conditions to DNA set forth in any one of (K) to (N);
(6) a single-chain antibody capable of binding to hepatitis B virus core protein, the antibody being encoded by DNA set forth in any one of the above (1) to (5);
(7) a vector containing DNA set forth in any one of the above (1) to (5);
(8) a transformant transformed with a vector of the above (7);
(9) a process for producing a single-chain antibody capable of binding hepatitis B virus core protein, the process being characterized in that a transformant of the above (8) is cultured under conditions which allow expression of the single-chain antibody encoded by DNA set forth in any one of the above (1) to (5);
(10) a therapeutic agent for hepatitis B comprising a single-chain antibody of the above (6) as an active ingredient;
(11) a gene therapy agent against hepatitis B comprising DNA set forth in any one of the above (1) to (5) as an active ingredient;
(12) a gene therapy agent of the above (11) characterized in that it uses an adenoviral vector; and
(13) a gene therapy agent of the above (11) or (12) wherein hepatitis B is acute or chronic hepatitis.
The present invention is described in detail below.
The DNA of the present invention is characterized in that it encodes a single-chain antibody capable of binding to hepatitis B virus core protein. In general, a single-chain antibody is composed of a contiguous peptide in which an appropriate linker sequence encoding a highly flexible peptide sequence is sandwiched between VH and VL moieties of an antibody.
Specific examples are the following 1) to 3):
1) a single-chain antibody which comprises VH moiety consisting of the amino acid sequence shown in SEQ ID NO: 4 or another moiety functionally equivalent to said VH moiety and which is capable of binding to hepatitis B virus core protein;
2) a single-chain antibody which comprises VL moiety consisting of the amino acid sequence shown in SEQ ID NO: 6 or another moiety functionally equivalent to said VL moiety and which is capable of binding to hepatitis B virus core protein; and
3) a single-chain antibody which develops its ability to bind to hepatitis B virus core protein by combining VH moiety consisting of the amino acid sequence shown in SEQ ID NO: 4 or another moiety functionally equivalent to said VH moiety with VL moiety consisting of the amino acid sequence shown in SEQ ID NO: 6 or another moiety functionally equivalent to said VL moiety.
A DNA encoding a single-chain antibody which meets the requirements of the above 1) may be, for example, DNA which comprises a DNA selected from the group consisting of:
(A) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4,
(B) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 4,
(C) DNA which comprises the base sequence shown in SEQ ID NO: 3,
(D) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 3, and
(E) DNA which hybridizes under stringent conditions to DNA set forth in any one of (A) to (D); and which encodes a single-chain antibody capable of binding to hepatitis B virus core protein.
Since the base sequence shown in SEQ ID NO: 3 is that of DNA encoding VH moiety of an antibody against hepatitis B virus core protein, and the amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence deduced from the base sequence shown in SEQ ID NO: 3, DNAs of the above (A) to (E) are those DNAs that encode VH moiety of an antibody against hepatitis B virus core protein or another polypeptide functionally equivalent to said VH moiety.
Likewise, a DNA encoding a single-chain antibody which meets the requirements of the above 2) may be, for example, DNA which comprises a DNA selected from the group consisting of:
(F) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6,
(G) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 6,
(H) DNA which comprises the base sequence shown in SEQ ID NO: 5,
(I) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5, and
(J) DNA which hybridizes under stringent conditions to DNA set forth in any one of (F) to (I); and which encodes a single-chain antibody capable of binding to hepatitis B virus core protein.
Since the base sequence shown in SEQ ID NO: 5 is that of DNA encoding VL moiety of an antibody against hepatitis B virus core protein, and the amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence deduced from the base sequence shown in SEQ ID NO: 5, DNAs of the above (F) to (J) are those DNAs that encode VL moiety of an antibody against hepatitis B virus core protein or another polypeptide functionally equivalent to said VL moiety.
Furthermore, a DNA encoding a single-chain antibody which meets the requirements of the above 3) may be, for example, DNA which comprises both of a DNA selected from the group consisting of the following (A) to (E) and a DNA selected from the group consisting of the following (F) to (J), and which encodes a single-chain antibody capable of binding to hepatitis B virus core protein:
(A) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4,
(B) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 4,
(C) DNA which comprises the base sequence shown in SEQ ID NO: 3,
(D) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 3,
(E) DNA which hybridizes under stringent conditions to DNA set forth in any one of (A) to (D),
(F) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6,
(G) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 6,
(H) DNA which comprises the base sequence shown in SEQ ID NO: 5,
(I) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5, and
(J) DNA which hybridizes under stringent conditions to DNA set forth in any one of (F) to (I).
Such DNA contains both of a DNA encoding VH moiety of an antibody against hepatitis B virus core protein or another polypeptide functionally equivalent to said VH moiety and a DNA encoding VL moiety of said antibody or another polypeptide functionally equivalent to said VL moiety. Such DNAs which meet the requirements of 3) are more preferable because they encode antibodies that specifically bind to only hepatitis B virus core protein.
Further examples of DNA which meets the requirements of 3) may be those DNAs which have a sequence selected from the group consisting of:
(K) DNA which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2,
(L) DNA which encodes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2,
(M) DNA which comprises the base sequence shown in SEQ ID NO: 1,
(N) DNA which comprises a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 1, and
(O) DNA which hybridizes under stringent conditions to DNA set forth in any one of (K) to (N), and which encode a single-chain antibody that binds to hepatitis B virus core protein.
In this connection, the base sequence shown in SEQ ID NO: 1 is the base sequence of DNA encoding PRE-HV, VH moiety of an antibody against hepatitis B virus core protein, linker, VL moiety of said antibody, and TAIL, and the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence deduced from the base sequence shown in SEQ ID NO: 1.
An example of the above DNA that hybridizes under stringent conditions is DNA hybridizing under the conditions in which hybridization is conducted overnight at 42xc2x0 C. using, as a hybridization buffer, a solution having the composition of 0.1% SDS, 50% formamide, 5xc3x97SSC, 1xc3x97Denhardt""s reagent, and 250 xcexcg/mL salmon sperm DNA, subsequently washed with 2xc3x97SSC for 1 hour at room temperature, with 2xc3x97SSC, 0.1% SDS for 30 minutes at room temperature, and then with 0.1xc3x97SSC, 0.1% SDS for 30 minutes at 50-65xc2x0 C.
Although the method for introducing deletion, substitution, insertion, or addition of bases or amino acids as described above is not specifically restricted, it can be achieved by introducing a mutation into the objective base sequence using a method which uses restriction enzyme, nuclease, or the like, or using site-directed mutagenesis (W. ITO et al., Gene, 102, 67-70 (1991), incorporating the product into a vector for expression, and transforming host cells with that vector. Furthermore, although the term xe2x80x9cseveralxe2x80x9d is not specifically restricted in connection with the above mutation of base or amino acid, it refers to such a number that permits the deletion, substitution, insertion, or addition by well-known methods such as site-specific mutagenesis described above.
Likewise, the amino acid sequences of the peptides corresponding to PRE-HV, linker, and TAIL in the single-chain antibody, and the base sequences of DNAs encoding said peptides are not specifically restricted, and they may have the amino acid sequences of peptides at corresponding sites in known single-chain antibodies and the base sequences of DNAs encoding said peptides. For example, when a single-chain antibody is prepared using Recombinant Phage Antibody System manufactured by Pharmacia, the amino acid sequences of the peptides corresponding to PRE-HV, linker, and TAIL are those sequence shown in SEQ ID NOs: 8, 10, and 12, respectively, and the base sequences of DNAs encoding said peptides are those sequences shown in SEQ ID NOs 7, 9, and 11, respectively.
In the present invention, DNAs of the present invention can easily be obtained by using a kit for preparation of single-chain antibody such as Recombinant Phage Antibody System manufactured by Pharmacia. Since DNA of the present invention prepared using such a kit is obtained in the form that is incorporated in a vector, expression of the single-chain antibody may be achieved more easily.
A method for preparing DNA of single-chain antibody of the present invention is described in detail below. An anti-hepatitis B virus core protein antibody can be produced in an animal by immunizing the animal with a mixture of hepatitis B virus core protein as the antigen, and an adjuvant.
The animal to be immunized is not specifically restricted, and may be, for example, rabbit, guinea pig, goat, donkey, fowl, rat, and mouse (Balb/c mouse, SWISS 3T3 mouse, C3H mouse, C57BL/6 mouse, and the like).
The hepatitis B virus core protein used as the antigen may be obtained, for example, by gene recombinant techniques (J. Electron Microsc., (Tokyo), 43(6), 386-393 (1994)).
The adjuvant is not specifically restricted, and may be a known adjuvant commonly used, for example, Freund""s complete or incomplete adjuvant.
According to the usual method, cDNA may be obtained by purifying poly(A)-RNA from the spleen of the immunized animal, preparing mRNA, and reverse-transcribing such mRNA.
Although the method for obtaining DNAs encoding VH and VL moieties of an antibody against hepatitis B virus core protein from such cDNA is not specifically restricted, they may be obtained, for example, from cDNA library by hybridization using nucleic acid probes specific for VH and VL moieties, respectively, or by specifically amplifying VH and VL moieties using PCR method.
When PCR method is used, for example, cDNAs encoding VH and VL moieties, respectively, can be obtained by conducting PCR with primer pairs specific for VH and VL moieties, respectively, under conditions that allows specific amplification using the above cDNA as template. As such a specific primer pair, those primers included in the above-mentioned Recombinant Phage Antibody System may be used.
The cDNAs for VH or VL moieties obtained may be each converted into double-stranded cDNA, and they may be ligated to a vector such as a phagemid vector after blunting the termini or after introducing restriction enzyme sites using a method such as PCR or site-specific mutagenesis and treating with restriction enzymes.
For example, the respective cDNAs for VH and VL moieties obtained as described above are ligated in a linear from to a DNA encoding a liker region, and restriction enzyme sites are further introduced into the contiguous linear DNA having the structure of VH-linker-VL by conducting PCR using a primer pair corresponding to the contiguous linear DNA having the structure of VH-linker-VL each of which primers has a restriction enzyme recognition sequence at its 5xe2x80x2 end in order to facilitate the ligation to a vector.
In this process, it is preferable to use a vector which has been constructed so that the DNA encoding PRE-HV and the DNA encoding TAIL will be connected to the ends of DNA encoding VH moiety-linker-DNA encoding VL moiety. An example of such vector is the phagemid vector pCANTAB5E included in Recombinant Phage Antibody System manufactured by Pharmacia.
The DNA of the present invention obtained by using such a kit is in the form that is incorporated in a vector. Therefore, a recombinant phage displaying a single-chain antibody can be obtained by transforming E. coli with said vector and then infecting the obtained E. coli clone with a helper phage.
A single-chain antibody of the present invention is a protein encoded by DNA of the present invention which fulfills one of the above-described requirements 1), 2) and 3).
One can determine whether or not the single-chain antibody described in the present specification is capable of binding to hepatitis B virus core protein, using ELISA by measurement of fluorescence intensity. In this procedure, a single-chain antibody of which fluorescence intensity is clearly higher than that of a negative control using a single-chain antibody which does not bind to hepatitis B virus core protein, and is reproducibly detectable is considered as a single-chain antibody capable of binding to hepatitis B virus core protein.
Vectors of the present invention are those vectors that contain the above-described DNA of the present invention. A vector used in constructing vectors of the present invention is not specifically restricted so long as it is a vector that allows expression of the single-chain antibody encoded by said DNA. A specific example is a phagemid vector pCANTAB5E (Pharmacia) when the host is E. coli, or pZeoSV2 (Invitrogen) when the host is a mammalian cell. The method for inserting the DNA of the present invention into the vector is not specifically restricted, and may be a known method, for example, using T4 DNA ligase.
Transformants of the present invention can be obtained by introducing a vector of the present invention into desired host cells.
Host cells are not specifically restricted, and include, for example, human cells such as hepatoma-derived HepG2, Chang liver cells, and kidney-derived adenovirus EIA and EIB transformed cells (293), animal cells such as COS-1, COS-7, CHO, and NIH 3T3, insect cells such as armyworm-derived Sf9 cells, yeasts, and E. coli such as E. coli strain K12 derivatives. For the purpose of expressing a human-derived single-chain antibody against HBV, it is more preferred to use human-derived cultured cells.
In order to introduce a vector containing DNA of the present invention into host cells, such a method as the calcium phosphate method, electroporation, lipofection, or recombinant viral infection may be used.
A process of the present invention for producing a single-chain antibody capable of binding to hepatitis B virus core protein is characterized in that a transformant of the present invention is cultured under conditions that allow expression of the single-chain antibody encoded by DNA of the present invention. As the conditions that allow expression of the gene product encoding a single-chain antibody that has been incorporated into the vector containing the DNA of the present invention, it is required that transcription from a promoter in the vector located upstream to the DNA of the present invention works effectively, and that an open reading frame for correct amino acids must can be assigned in the translation.
Expression of single-chain antibody of the present invention can be confirmed using a method as described above by measuring the ability of the obtained single-chain antibody to bind to hepatitis B virus core protein.
Purification of anti-core protein single-chain antibody from the recombinant may be achieved by a conventional known method. For example, cultured cells may be broken and the desired single-chain antibody expressed may be obtained from the supernatant, for example, by affinity column chromatography. Specifically, in the present invention, the antibody can easily be purified by affinity column chromatography against E-tag at the C-terminus of the expressed anti-core protein single-chain antibody. The anti-core protein single-chain antibody obtained as described above has an ability to suppress DNA synthesis of the virus by binding to hepatitis B virus core protein, and can further achieve the effect of suppressing propagation of the virus.
A therapeutic agent for hepatitis B of the present invention may be used as a therapeutic agent that comprises as an active ingredient an anti-core protein single-chain antibody of the present invention as such, or may be used as a gene therapy agent by which DNA encoding an anti-core protein single-chain antibody is administered into a patient as an active ingredient of the pharmaceutical agent and the gene is expressed in the body of the patient.
A therapeutic agent that comprises as an active ingredient an anti-core protein single-chain antibody as such may be administered with an adjuvant or administered in a particulate dosage form. More particularly, the dosage forms such as liposome formulations, particulate formulations in which the active ingredient is attached to beads having a diameter of several xcexcm, and formulations in which lipids are attached to the ingredient may be used. Administration may be achieved, for example, by means of sustained-release mini-pellet formulations. Furthermore, peptides that enhance the efficiency with which the antibody is transferred across the cell membrane may also be added. In particular, hydrophobic regions are connected to the N- and C-termini of the single-chain antibody. It is also possible to enhance the permeability of the cell membrane by attaching a particular carbohydrate chain. Although the amount administered may be adjusted as appropriate depending on the amount of hepatitis B virus carried by the patient, the age, weight of the patient, and the like, it is typically 0.001 mg/kg/dose to 1000 mg/kg/dose administered for consecutive days at the beginning and then administered once every several days or several months. Regarding the mode of administration, the agent can be administered orally, intraarterially, intravenously, or intramuscularly depending on the dosage form.
When used as a gene therapy agent, the present agent can highly express an anti-core protein single-chain antibody intracellularly, thereby suppress replication of the viral DNA in cells infected with hepatitis B virus, and further suppress propagation of the virus.
In the case that a gene therapy agent comprising as an active ingredient DNA encoding an anti-core protein single-chain antibody is introduced into infected cells, the modes of administration are broadly divided into two kinds of embodiments, one using non-viral vector and the other using viral vector. In particular, representative examples of the former embodiments are those methods in which a DNA molecule of the present invention is directly introduced into cells (such as a method in which an expression plasmid is directly administered intramuscularly), those methods in which the DNA molecule is introduced using liposome (such as the liposome method or the lipofectin method), those methods in which the DNA molecule is transferred into cells together with a carrier using a particle gun, microinjection, the calcium phosphate method, and electroporation.
Representative examples of the latter embodiments are those methods that use viral vectors such as recombinant adenoviruses or retroviruses, and in particular, include those methods in which DNA encoding an anti-core protein single-chain antibody is incorporated into a DNA or RNA virus such as nontoxic retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, or Sendai virus, and introduced into cells.
Among these viruses, adenovirus is known to have a very high transferability into the liver tissue, and also to exhibit an infection efficiency much higher than other viral vectors. It is therefore preferred to use an adenoviral vector system.
By using one of these methods, an anti-core protein single-chain antibody gene can be introduced into hepatitis B virus-infected cells. Furthermore, in the case of gene therapies using non-viral vectors, it is desired to guide the gene to the vicinity of infected cells, for example, by local administration, whereas in the case of gene therapies using viral vectors, it is not necessarily required to locally administer the agent, and intravenous administration is also possible.
As methods by which a gene therapy agent of the present invention is allowed to act as a pharmaceutical in practice, there are an in vivo method in which DNA is directly introduced into the body, and an ex vivo method in which certain cells are removed from a human, DNA is introduced into said cells ex vivo, and the cells are returned into the body (Nikkei Science, April, 1994, pp. 20-45 and September, 1997, pp. 27-62; Gekkan-Yakuji, 36 (1), 23-48 (1994); and Jikkenn-Igaku, 12 (15), 1994). In the present invention, an in vivo method is more preferred. Although the content of DNA in the formulation may be adjusted as appropriate depending on the disease to be treated, the age, weight of the patient, and the like, it is typically 0.0001-100 mg, and preferably 0.001-10 mg, administered once every several days or several months.
The therapeutic agents for hepatitis B comprising as an active ingredient an anti-core protein single-chain antibody, and the gene therapy agents against hepatitis B comprising, as an active ingredient, a DNA encoding an anti-core protein single-chain antibody are those agents against hepatitis B caused by hepatitis B virus, and said hepatitis B may be acute or chronic hepatitis.